The major aim of this project was to attempt to define residues In rlcln A chain which are involved In the catalytic activity of the protein and to define a rlcin A chain molecule of minimum size which still remains active. A simple and sensitive system was developed in which the expression and assessment of biological activity of recombinant rlcln A chain are combined. This represents one of few reported examples of the ability to assess the activity of protein expressed from in vitro synthesised RHA in a cell free system. When recombinant ricln A chain transcripts were translated in a rabbit reticulocyte lysate, the ribosomes were rapidly inactivated. In contrast, ribosomes which have translated transcripts encoding non toxic polypeptides such as ricin B chain are not inactivated. Ribosome inactivation was accompanied by a highly specific modification of 28S rRNA which Is thought to cause the inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under conditions which inhibit reticulocyte ribosomes, confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inactivation by ricln A chain than are mammalian ribosomes. Using the same system, it was shown that by deleting an internal hexapeptide which shares homology with hamster EF 2, catalytic activity was completely abolished. Deleting a second Internal pentapeptide, conserved between ricin A chain and trichosanthln, had no effect. Deleting the first nine residues from the V terminus of ricln A chain did not affect toxicity, whereas deleting a further three residues Inactivated the polypeptide. Point mutations which Individually converted arginine 48 and arginine 56 to alanine residues or which removed arginine 56 were also without effect on the catalytic activity of the toxin.

The circadian clock is an endogenous mechanism that provides a wide variety of organisms with the ability to anticipate daily environmental changes. It was shown that under cyclic and constant light growth conditions Arabidopsis exhibits rhythmicity in Botrytis cinerea resistance, with maximal resistance observed when leaves were inoculated at dawn. Crucially, this mechanism was confirmed to be under circadian clock regulation. To understand how the circadian clock was driving an effective defence response, genes that were more rapidly induced or repressed after inoculation at dawn compared to night were identified. This indicated a complex interaction between the circadian clock and the defence regulatory network. Phytohormone defence signalling, in particular, jasmonate (JA) and ethylene responses, was shown to contribute to the observed rhythmic variation in resistance. This was further confirmed by the identification of a JA signalling mutant (jaz6), which displayed no difference in resistance to B. cinerea following inoculation at dawn or night under cyclic or constant light conditions. Given the central role of JAZ6 in the circadian defence response against B. cinerea, it was likely transcription factors (TFs) bound by JAZ6 were potential links between the plant circadian clock and the defence response. Elucidating the TFs that interacted with JAZ6 revealed JAZ6 to be able to interact with a TF shown to be crucial to the B. cinerea defence response, EIN3. Moreover, JAZ6 was also able to interact with a central regulator of circadian clock, FHY3. Both TF interactors indicate JAZ6 is a linking protein between the circadian clock and the defence response against B. cinerea. To further understand how the circadian clock was mediating the plant defence response against B. cinerea genome-wide chromatin accessibility data was generated using ATAC-Seq. This aimed to enable the comparison of chromatin accessibility as well as TF binding in regions surrounding genes related to B. cinerea defences between the two time-points. This protocol was not optimized for plant tissue. Steps within in! laboratory-based ATAC-Seq protocol were therefore pinpointed for tissue specific optimization. Thus, a protocol specific to ATAC-Seq data analysis was proposed.

Plants are exposed to a variety of abiotic stresses, including salinity and drought. These environmental stresses cause major losses in crop yield. High salinity stress alone impairs crop production on at least 20% of irrigated land worldwide. Thus, the development of stress-tolerant crops is of major importance for food security. Many physiological responses to ensure acclimation to adverse environmental conditions require the synthesis and perception of the plant hormone abscisic acid (ABA). Recent studies have shown that the function of the circadian clock is altered under some abiotic stress conditions such as drought, and osmotic stress. The first part of this thesis investigates the role of the stress response hormone abscisic acid in changing the function of the clock under osmotic stress. It was found that multiple core clock genes are responsive to ABA application, with sharp transient induction of morning associated genes in particular. In comparison, osmotic stress caused a damping of the amplitude of gene expression. It was then shown that the disruptive effect of osmotic stress on circadian leaf movement rhythms required the biosynthesis of ABA. This is important as it demonstrates that ABA is a key factor in mediating osmotic stress responses to the clock. The second half of this thesis then focuses on how altered function of the clock might impact plant performance under drought or osmotic stress. It was found that the morning associated LATE ELONGATED HYPOCOTYL (LHY) transcription factor, which functions as a key component of the circadian clock, regulates many of the components of the ABA signalling pathway. Evidence was provided that, while overexpression of LHY results in reduced ABA levels, ABA responsive gene expression is significantly increased upon ABA treatment. Finally, through phenotypic analysis it was determined that increased LHY expression leads to increased performance in drought and osmotic stress conditions. This is important as it suggests that manipulation of circadian clock function may be useful as a novel approach in the future engineering of stress tolerant crop lines.

White blister rust caused by the obligate pathogen Albugo candida is infectious across the Brassicaceae, and is an economically important disease of cultivated Brassica species. The advance in genotyping technologies has made possible the understanding and deployment of host resistance to plant pathogens, previously unachievable through conventional plant breeding. In this work, the application of Genotyping by Sequencing (GBS) and Resistant Gene Enrichment Sequencing (RenSeq) has identified a single GDSL lipase as a candidate for recessive race non-specific resistance to A. candida in B. oleracea. A second locus has been identified conferring dominant race specific resistance to an A. candida isolate collected from Australia. Much work has been achieved in understanding the genetic basis of resistance to A. candida in the model organism Arabidopsis thaliana, including the identification of white rust resistance (WRR)4, a single dominant resistance (R) gene conferring resistance to A. candida races 2, 4, 7 and 9 in A. thaliana Columbia. In this thesis research, three Columbia-virulent isolates were characterised that are capable of breaking WRR4-Col mediated resistance. Two of these were used to map a new broad spectrum resistance locus, designated WRR4-OyC1, in the vicinity of WRR4 in the Norwegian A. thaliana accession Oy-0 and two additional minor effect QTLs. All three isolates were used for association genetic analysis of genome-wide ‘effectorome’ sequencing to identify candidate genes for avrWRR4 in A. candida for both WRR4, and Oy-0 recognition. From the combined results of this research, a potential strategy for durable white rust control in oilseed and vegetable Brassica would be stacking of at least two R alleles (WRR4-Col and WRR-OyC1) in a genetic background containing the recessive, resistance allele of the GDSL lipase.

Plants establish associations with beneficial fungi in order to overcome abiotic and biotic stresses. An example is Serendipita indica, which colonises the roots of a broad range of plant species, including Arabidopsis thaliana. S. indica has been shown to increase crop yield, induce tolerance against drought and salinity, and enhance pathogen resistance in plants. However, the underlying molecular mechanism, utilised by S. indica to confer these beneficial effects to the host plant, is unknown. S. indica may induce beneficial effects in plants, through the use of effector proteins. Sequencing of the S. indica genome, revealed 386 putative effectors. To elucidate the full sequences of these effector candidates, the S. indica transcriptome was sequenced. A shortlist of 150 S. indica effector candidates was generated for experimental investigation. The potential A. thaliana targets of these 150 putative effectors, were identified through Yeast-two hybrid screening, and by identifying differentially regulated genes from RNA-seq data. Only 38 A. thaliana proteins, that were targeted by the S. indica effector candidates, are known pathogen effector targets. Therefore, the majority of S. indica plant targets are novel, and may not be affected by plant pathogens. These novel targets provide new opportunities, for scientists to engineer crop plants that produce greater yields, and exhibit enhanced resistance to drought, salinity and disease. One pathogen S. indica is known to induce resistance against, is the necrotrophic pathogen Fusarium graminearum. To identify if any S. indica effector candidates, were responsible for protecting barley from F. graminearum, a screening process was set up. This involved the optimisation of a stable barley root transformation system, and the adaptation of F. graminearum inoculation procedures. A metabolite extraction procedure was also developed, to determine if any S. indica specialised metabolites were involved in promoting F. graminearum resistance in barley.

Parsnips (Pastinaca sativa) are a speciality crop, covering 3000 hectares across the UK, with a 93,000-tonne production and economic value of greater than £31M annually. Currently, the major constraints to production are losses associated with root canker disease, caused by a range of fungal pathogens. With no specific fungicides, development of long-term, sustainable resistance to parsnip canker is highly desirable. This work characterises the pathogens responsible, and develops tools to facilitate breeding for quantitative resistance to root canker diseases. Isolations and molecular characterisation of pathogens responsible for parsnip canker highlighted a range of fungal species, whilst canker symptoms were found to be clearly associated with certain pathogens. Cylindrocarpon destructans, Mycocentrospora acerina and to a lesser extent Itersonilia pastinacae were identified as the primary pathogens responsible for causing parsnip canker in the UK. Itersonilia spp. isolates from a range of hosts were found to infect parsnip roots and leaves, and produce both chlamydospores and ballistospores at a range of temperatures; furthermore, molecular characterisation failed to differentiate between species. For these reasons, Itersonilia should be described as a single species. For both C. destructans and M. acerina, isolates showed minimal variation in pathogenicity on parsnip roots and seedlings, and exhibited mycelial growth even at low temperatures. Phylogenetic analysis identified a species complex for both pathogens that could not be resolved by the ITS (Internal transcribed spacer) alone. Finally, parsnip root and seedling assays were developed to determine resistance to I. pastinacae, M. acerina and C. destructans within parsnip populations. QTL analysis of a parsnip genotyping population identified a significant QTL conferring resistance to M. acerina for use in a marker assisted breeding programme. The understanding of the pathology gained in this project will facilitate selection of resistant varieties, benefitting breeders, growers and through reduction in control mechanisms, society in general.

This research establishes molecular breeding capability for adapting common bean (Phaseolus vulgaris L.) to UK growing conditions. A high-resolution linkage map was constructed for a bi-parental recombinant inbred population (large brown x small white haricot) using genotyping-by-sequencing data. Pre-breeding material was exploited to enable genetic mapping and marker-assisted selection of essential adaptive traits, including (1) resistance to halo blight, caused by Pseudomonas syringae pathovar phaseolicola (Psph), (2) root architecture related to abiotic stress tolerance and nutrient acquisition, (3) earliness of maturity, (4) plant architecture amenable to mechanical harvest, and (5) seed coat colour of consumer interest. A 500-kb mapping interval was defined for quantitative resistance to the broadly virulent Psph race 6, a devastating bacterial pathogen that threatens global bean production with losses from halo blight. Complementary research generating high-quality draft genomes for 32 pathogenically and geographically diverse isolates of Psph identified five high-probability candidate determinants of the broad virulence of Psph race 6, including avirulence protein AvrD. Pathogenicity effectors that are highly conserved within the pathovar were identified as candidate targets for potential race-nonspecific resistance to halo blight. Putative QTL for root architecture traits associated with water and nutrient acquisition were detected on chromosome Pv07. A useful breeding strategy may be to select for larger taproot diameter in view of the comparatively high heritability of this trait. Potentially desirable alleles on Pv07 are linked in coupling phase with the dominant allele of seed coat pigmentation factor P. Identification of lines recombinant for these alleles may prove useful for the introgression of genes governing physiological resilience into white-seeded varieties adapted to UK growing conditions. Provisional QTL for morphological and reproductive traits of agronomic importance, including plant architecture, growth stage and yield, were identified using phenotypic data obtained from pilot field and polytunnel evaluations of the recombinant inbred population.

Glutamine synthetase (GS) catalyses the assimilation of ammonia with glutamate, to form glutamine. In higher plants GS is an octameric enzyme of Mr 360,000 located in plastids and the cytosol. In the legume Phaseolus vulgaris L., GS is encoded by four nucleur genes (g1n-α, g1n-β, g1n-γ and g1n-δ) which encode the cytosolic α, β, and γ GS subunits of Mr 39,000, and the plastid δ GS polypeptides of Mr 44,000, accordingly. The aim of the work is to study the regulation of expression and structure of P. vulgaris root nodule GS. This thesis has reported the construction of a P. vulgaris nodule cDNA library, and the isolation and characterisation of a full length nodule GS cDNA clone. The polypeptide encoded by this cDNA and two previously identified GS cDNA clones, pR-1 and pR-2 (see Gebhardt et al, 1986, EMBO J. 5, 1429-1435) have been produced in vitro by transcription/translation and shown to co-migrate on two-dimensional gels with the cytosolic α, β, and γ GS polypeptides respectively. An RNase protection technique has been used to specifically and quantitatively determine the abundance of the g1n-β, g1n-γ and leghaemoglobin (Lhb) mRNAs. Differences in the relative abundances of g1n-β and g1n-γ mRNAs at different stages of nodulation suggest that these two genes are divergently regulated, whereas the g1n-γ and Lhb mRNAs are coordinately expressed during nodule development, detectable at least one day prior to the onset of dinitrogen fixation. Furthermore, the detection of the γ polypeptide, although at a reduced level, in nodules grown in the absence of dinitrogen (under an atmosphere of 80% Argon: 20% oxygen) suggests that a product of dinitrogen fixation does not provide the primary signal for, but may have a role in the level of, g1n-γ nodule expression. A study of the abundance of the g1n-γ and Lhb mRNAs from a variety of P. vulgaris organs has identified that in contrast with the nodule specific expression of Lhb mRNA, g1n-γ mRNA is also detectable, albeit at a lower abundance, in stems, petioles, and green cotyledons. The structure of P. vulgaris nodule and plumule GS has been studied through the resolution of the component GS isoforms, and the determination of their respective GS polypeptide compositions by ion exchange fast protein liquid chromatography and two-dimensional western blotting respectively. These studies have identified that both α + β, and β + γ GS subunits may assemble, perhaps randomly, to produce a heterogenous mixture of GS isoforms. However, a study of the changes in GS isoforms during nodulation has shown that the β8 isoform is present at higher activity than would be expected, which could reflect the differences in the temporal and/or spatial expression of the γ and β GS subunits within the nodule. The three cytosolic GS cDNAs have been expressed individually In Escherichia coli, synthesising GS subunits of the correct Mr, which appear for α, γ and β to be mainly soluble, intermediate in solubility and insoluble respectively. The soluble α, β and γ GS subunits are able to assemble into kinetically active oligameric isoenzymes, in the absence of any specific plant assembly factors. In addition, the α and γ cDNAs have been shown to complement an E.coli g1nA mutation, suggesting that the recombinant α and γ higher plant GS enzymes can effectively function in the E.coli nitrogen assimilatory pathway.

Three proteins located in the thylakoid lumen, plastocyanin and the 23KDa and 33KDa oxygen evolving polypeptides of photosystem II, are synthesised in the cytoplasm as higher molecular weight precursors, with N-terminal transit peptides. Import of these proteins involves removal of the first part of the transit peptide by a stromal processing peptidase to yield an intermediate. Maturation, by removal of the remaining transit peptide is performed by a thylakoidal processing peptidase (TPP). TPP has been partially purified and characterised from pea thylakoids and found to be an integral thylakoid membrane protein with the active site on the lumenal (trans) side of the membrane. TPP has a molecular weight of less than 250 000 and is not associated with any supra-molecular complex. Partial purification has yielded ten bands on a Coomassie stained SDS-PAGE gel; however TPP has not been attributed to any of these bands. TPP displays specificity for chloroplast protein precursors with transit peptides containing a thylakoid transfer domain; however, no species specificity is displayed. TPP exhibits similarities in reaction specificity to Escherichia coli leader peptidase (LEP) in that both peptidases cleave the same eukaryotic and bacterial precursor as well as cleaving higher plant lumenal precursors at the predicted cleavage site. No standard protease inhibitor has been found to abolish TPP activity; however, a synthetic signal sequence polypeptide will inhibit TPP and LEP. A thylakoidal endopeptidase (EPS) has been discovered which cleaves lumenal precursors to a size slightly larger than the mature size. EP5 displays different inhibitor sensitivities to TPP and is either a high molecular weight protein or associated with a supramolecular complex. EPS is assumed to be involved in the turnover of thylakoid proteins.

The synthesis of the small subunit of ribulose-1,5-bisphosphate carboxylase from leaves of pea (Pisum sativum) and its assembly into the holoenzyme have been reconstituted in cell-free extracts. The small subunit precursor has been synthesised by translating its messenger RNA in a cell-free protein-synthesising extract derived from wheat-germ. This precursor has been processed by a soluble extract from isolated chloroplasts, and the processed molecule shown to assemble into the holoenzyme. Features of the synthesis and processing of this polypeptide are discussed both in relation to the synthesis of other proteins which are transported across membranes, and in relation to chloroplast development. Synthetic DNA molecules encoding the messenger RNA for the small subunit precursor have been. Isolated by molecular cloning in a plasmid of Escher ich ia coli. These cloned molecules have been employed to characterise, and to purify, the messenger RNA of the small subunit precursor. The cloned DNA molecules have also been employed to probe pea leaf nuclear RNA for complementary sequences. These studies suggest that the messenger RNA may be first synthesised as a precursor of nearly twice the final size. In etiolated pea leaves, where rlbulose-1,5-bIsphosphate carboxylase is present In reduced amounts relative to green tissue, the messenger RNA for the small subunit precursor has also been shown to be present in reduced amounts. Furthermore nuclei isolated from etiolated tissue contain reduced amounts of transcripts of the small subunit gene. These results suggest that the expression of the gene for the small subunit of rlbulose-1,5-bisphosphate carboxylase may be controlled at the level of transcription.